KR910006811B1 - Process for preparing high purity dermatan sulfate - Google Patents

Abstract

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Description

[Name of invention]

Process for the preparation of high-purity dermatan sulfate

Detailed description of the invention

The present invention relates to a novel process for preparing high-purity dermatan sulphate and to pharmaceutical compositions containing the same as active ingredients.

Dermatan sulfate (DS) is a mucopolysaccharide (MPS) consisting of repeating units of disaccharides of the following structural formula containing 1 mole of iduronic acid and 1 mole of N-acetyl galactosamine sulfonate, linked by 1,3 glucoside bonds Known as

The molecular weight of DS can vary depending on the method used to extract the proteoglycans and the method used to separate it from MPS.

The ratio of carboxyl to sulfonic acid group is theoretically 1: 1, which in practice may also change due to the presence of a large number of sulfonated hydroxyls in some galactosamine and iduronic acid units.

It has recently been found that DS can show significant antithrombin action without interfering with many other serine protease inhibitors that selectively activate heparin cofactor II to regulate the blood coagulation process.

However, its use as a medicament does not lead to the chemical alteration or degradation of the various groups present, and to the possibility of its extraction from natural proteoglycans in an unaltered state, as well as to foreign substances (eg proteins and nucleotides) that can cause negative side effects. ) Is actually determined by the possibility of completely removing it.

In practical terms, it has proved very difficult to establish a DS manufacturing method that satisfies the above requirements.

Therefore, a study of extracting rat skin proteoglycans with an inorganic salt solution at varying concentrations (Chemical Abstracts-vol, 84, 1976-page 194-abstract 71173d) resulted in a mixture of other proteoglycans with a small amount of dermatansulfate. Was obtained. Separation of pure dermatan sulfate from a mixture of glycosaminoglycans is difficult to carry out and results in a very low yield of the final product.

Also, as described in EP 0097625, dermatan sulfate obtained from aqueous solution of glycosaminoglycans by fractionation at different temperatures does not meet the desired purity.

Now, the present inventors have extracted the main problem of the present invention, that is, DS which is present in the starting material and does not contain any impure residues which are not actually changed in terms of branching and structure and may cause undesirable side effects. I found out how to make it.

The novel method essentially comprises the following steps: a) The raw material is preferably selected in the form of small intestine mucosa of bovine or swine, or in the form of bovine lung, pancreas, aorta, spleen, brain, thymus or cartilage, To prevent this, it is rapidly frozen: b) ultrafine micronized raw material homogenized with such or cold acetone with an aqueous solution of CaCl ₂ : c) an aqueous raw material / CaCl ₂ homogenate with proteolytic enzymes at alkaline pH and low temperature. Decompose with: d) acidify the digest, heat and filter: e) treat the clear filtrate with a quaternary ammonium salt capable of complexing MPS, and f) selectively filter DS with high purity.

The method of performing the individual manufacturing steps outlined is described in more detail as follows.

Step a) comprising the preparation of the raw material can be carried out in a variety of ways suitable to provide a suitable material for the next step.

Step b) comprising mixing with CaCl ₂ is one of the essential steps in the novel process and can be carried out in a variety of ways without changing the nature of the invention.

In the case of such a frozen homogeneous raw material, it is ultra-micronized using 0.01M CaCl ₂ aqueous solution in an amount such that the weight ratio of the raw material: solution is 1: 0.5.

When the material is used in the form of acetone-treated powder, the initial frozen material is ultra-micronized using an aqueous solution of 1M CaCl _{2 in} an amount such that the weight ratio of material: solution is 1: 1, and the mass is cold ( +5 ^o ) stirred with acetone at a weight ratio of 1: 3 and filtered, the residue was dissolved in cold acetone again at a weight ratio of 1: 2 and filtered, and the residue was once again dissolved in cold acetone at a weight ratio of 1: 1. The final residue may be dried under vacuum at 35 ° C. and stored at low temperature for subsequent enzymatic degradation, or introduced directly into the decomposition step by mixing in deionized water with a ratio of 1:20 in a suitable reactor.

Alternatively, the frozen starting material is ultramicronized using 0.1 M CaCl ₂ solution in an amount such that the weight ratio of material to solution is 1:10. The mixture is spray-dried with a small-bore rotor and air is introduced at a temperature not exceeding 150 ° C. with a contact time of no more than a few seconds. The powder formed in this way can be used directly by preserving it in a plastic bag at a low temperature for subsequent decomposition or by introducing it into the reactor using deionized water in an amount such that the weight ratio of powder to water is 1:20. have.

Lyophilization may be advantageous for small-scale processing, especially if the DS is extracted from delicate organs such as the thymus, pituitary gland or heart. In this case, the frozen material is ultra-micronized using a 1M CaCl ₂ aqueous solution containing 10% of powdered sorbitol having a weight ratio of raw material: CaCl ₂ solution of 1: 1.

After stratification to a thickness of 2 cm, it is frozen at −40 ° C. and lyophilized at 25 ° C. using a diffusion pump for residual moisture. The lyophilized product is in the form of a crumbly powder having a moisture content of about 2%. The lyophilized powder can be preserved for the next decomposition or diluted directly with deionized water in a ratio of 1:20 to carry out the next step.

Step c) comprising an enzymatic digestion step is performed with normal proteases (e.g. trypsin, chymotrypsin, and preferably alkalase, pastatase and superases) under conditions particularly moderated to prevent depolymerization or degradation of DS. It is characterized by performing using).

The aqueous mixture is preferably alkalined to pH 7-9 with CaOH ₂ and then heated to a temperature between 40 ° and 55 ° C. The enzyme is then added to the raw material in an amount such that the weight ratio of the raw material: enzyme is from 1: 0.0001 to 1: 0.001. Decomposition is continued for 6 to 24 hours while continuing the electrophoretic check to determine when proteoglycan degradation is complete. At this point, the mass is completely fluid.

The next step d) is carried out with the flowable digest, which may use a small amount of other acid, but to prevent the introduction of ions other than chlorine ions already present, preferably slightly acidic to a pH of 3-6 using HCI. And the fluidized mass is heated to 70 to 80 ° C. for about 1 hour.

The conditions of step d) lead to protein coagulation and precipitation of nucleoprotein derivatives in the form of complexes with Ca salts present. At the same time, pH and temperature conditions allow MPS to dissolve in the form of Ca salts. The solution is filtered through a rotary filter and, if possible, by a pressure filter to remove traces of salified fatty acids.

In this way an actually pure and completely clear filtrate containing only MPS in the form of Ca salts is obtained.

This clear solution can be used directly in the next step e), or preferably, it is first analyzed by electrophoresis to determine the quality and amount of MPS present.

As stated earlier, step e) consists essentially of treating with an ammonium salt which can form an insoluble complex with an aqueous solution MPS containing MPS Ca salt. However, it is an object of the present invention to obtain a DS of pharmaceutical purity, which can be used in two different ways: 1) dimethyl-ethyl-cetyl ammonium ethyl sulfate, which complexes the solution and selectively precipitates the DS Or 2) the solution is treated with a quaternary ammonium salt, preferably selected from hyamine, cetyl-trimethylammonium bromide and cetyl-dimethylethylammonium bromide, which can precipitate all MPS in complex form, From this, the DS complex is separated by selective solubilization with an organic solvent.

The choice of method depends primarily on the relative amounts of DS and MPS in the solution and whether or not the MPS is recovered.

In general, selective precipitation of DS according to method 1) is preferred.

To precipitate with dimethyl-ethyl-cetyl ammonium ethylsulfate, the salt is used in a quantity such that the weight ratio of the starting material is about 1: 1500 when the raw material is used in the form of a frozen homogenate, and the raw material is powdered. When used in the form of, add to the solution in an amount such that the weight ratio to raw material is 1: 300. The mixture is left for several hours. The DS complex precipitates rapidly because of the presence of Ca ions. Follow this and collect the complex by centrifuge.

However, if you want to precipitate all MPS according to method 2), raise the temperature of the transparent solution to 60 to 70 ℃, add the amount of ammonium salt equal to the weight of the electrophoretically measured MPS and dilute the mixture with deionized water. Lower the water concentration to 0.4M.

It is left for 5 to 12 hours and then centrifuged to recover the mixture of precipitated solid complexes. The mixture is washed with water to remove excess ammonium salt and dried.

The selective recovery of the high-purity DS by step f) is carried out differently depending on whether the method 1) or 2) is used for precipitation with the ammonium salt.

If the DS / dimethyl-ethyl-cetyl ammonium ethylsulfate complex is selectively precipitated and separated, the complex is treated with 2M CaCl ₂ aqueous solution containing 10% ethanol. The weight ratio of Ds complex to hydroalcoholic CaCl ₂ solution is 1: 5. The pH of the solution is adjusted to 7-9 with CaCH ₂ , heated to 60-80 ° C. for about 1 to 3 hours and then filtered.

The solution is introduced into a purification step, which is characterized by treatment in a 20 m ² or linear column with a molecular cut-off of 10,000, concentrated to 1: 10 and then diluted to 1: 1 The purification step is continued until the salt and remaining trace impurities are completely removed. DS was treated with acetone in an amount such that the volume ratio of solution: acetone was 1: 0.3, or with ethanol or methanol in an amount such that the volume ratio of solution: ethanol or methanol was 1: 0.5. Precipitate. Dermatan sulphate precipitate is in the form of a light powder.

The DS obtained by this method is preferably converted into an alkali salt by dissolving in a 2MNa, K, Li or Mg chloride solution in an amount such that the weight to salt solution volume ratio is 1:10. The mixture is stirred for 2-10 hours, diluted 1: 2 with distilled water, then filtered and precipitated using a 1: 0.5 ratio of acetone.

In this method, Na, K, Li or Mg salts of dermatan sulfate are obtained depending on the salt solution used.

When precipitation with ammonium salts is carried out according to method 2) of step e), the DS separation and purification of step f) is carried out in the following manner. The mixture of MPS ammonium salt complex is dispersed in acetone for about 1 to 2 hours at a temperature of about 25 ° C.

The DS complex is selectively dissolved in acetone, and the other complex remains in an undissolved state and separated by performing a separation treatment. The amount of acetone used is important to completely dissolve the DS complex. If it contains 10% DS complex of the treated mixture, add 1 volume of acetone (relative to the volume of the treated complex) and add 2 volumes of acetone if the mixture contains 20% of the DS complex. That is, always use the acetone volume according to the given DS content ratio.

The mixture treated with solvent is centrifuged or filtered. The clear acetone filtrate is treated with 3M salt solution (Na, K, Li or Mg chloride) at a volume ratio of acetone extract to salt solution of 0.25 to 1 at ambient temperature.

Under these conditions, the DS ammonium complex is separated and precipitated in the form of Na, K, Li or Mg salts depending on the salt solution used.

In all cases, the properties of the DS salts produced in step f) described herein are as follows.

Single bend for electrophoresis analysis in various buffer systems

Molecular weight: 20,000-40,000

Organic Sulfur: 6-8%

Uronic acid: 30-36%

Ratio of sulfate group to carboxyl group: 1.2 to 1.5

The ratio of uronic acid to galactosamine 1: 1

Iduronic acid: 80 to 90% of total uronic acid

-[α] _O : -60 ° to -65 °

Heparin activity: 10u / mg USP

In using the manufacturing method of the present invention, the DS yield varies depending on the original organ or tissue, and is 1 to 8 kg per 10,000 kg of raw material.

If it is desired to recover the MPS present in the starting material, the method used is preferably by method 2) of step e), ie total MPS precipitation as ammonium salt complex.

After dissolving the DS complex with acetone as described in the preceding step f), the undissolved mass was dispersed in 2 volumes of distilled water to remove the acetone residue, centrifuged and dissolved in ethanol following the method described for acetone. Suspend. Heparin-sulfate is separated in this way.

From the mass remaining insoluble in ethanol, the same method described for the other solvents is used and pure heparin is extracted by treatment with methanol.

The sodium, potassium, iridium, calcium or magnesium salts of dermatan sulfate obtained by the manufacturing process of the present invention are biologically active (about 60 μA IIa / mg) for activating the so-called heparin cofactor II, but partially thromboplastin activity. There is no anticoagulant action during the period and a weak factor Xa inhibitory activity (about 20 UA Xa / mg).

In vivo, the product is not toxic and does not cause bleeding, but has been shown to cause significant vasodilation. In addition, when administered intravenously, subcutaneously and ileum, it has an effect of inhibiting the formation of blood clots experimentally, and can partially protect animals from the damage caused by administration of adriamycin.

Topical administration of the product to diseases caused by the thrombosis of the superficial venous circulation shows rapid difference due to fibrinolysis and anti-inflammatory action.

DS obtained by the manufacturing process of the present invention is assessed zoologically and pharmacologically in experimental animals as described below.

[Acute Toxicity]

Acute toxicity is evaluated after oral, intravenous, intraperitoneal and subcutaneous administration of dermatan sulfate obtained by the manufacturing process of the present invention to Swiss mice.

The relative LD ₅₀ values are listed in Table 1, which shows that dermatan sulfate is low in acute toxicity.

TABLE 1

Toxicity of DS is assessed after repeated oral and intramuscular cold administration to Wistar rats for 5 weeks. The product is orally administered to different groups of mice daily at a dose of 200 mg / kg / day and intramuscularly administered at a dose of 20 to 40 mg / kg / day.

Two groups of mice treated with oral and intramuscularly administered physiological solutions, respectively, were used as controls.

During the treatment period, no changes in behavioral surface, weight or feed consumption were observed in rats treated with DS compared to rats treated with excipient alone.

After treatment, rats are anesthetized with ether. Mice treated with intramuscular administration showed no change at the time of injection. There were no significant changes in hematological, hemochemical or urinary parameters. Spleen weight was slightly increased in rats treated intramuscularly at a dose of 40 mg / kg, but histological examination revealed no damage to the spleen structure.

No further changes in organs were observed.

[Antithrombotic action]

hel, Breddin K., Zimmerrnan. Eds. pp 99, 1983]에 기재된 바와같이 쥐의 대정맥의 결찰에 정맥특성을 가진 혈전을 형성시킨다.The antithrombotic action of DS is evaluated by two experimental thrombosis models. First, Umetsu T., Sanaik., Thromb. Haemostas., 39, 1978, 74], the arteriovenous shunt of rats is mixed with the characteristics of veins and arteries to form thrombus. Second, Reyers et al., Standardization of animal models of thrombosis, 17th Angiological Symposium, Kitzb

hel, Breddin K., Zimmerrnan. Eds. pp 99, 1983 to form venous thrombosis in the ligation of the vena cava of rats.

Arteriovenous Shunt: Comparison of DS Antithrombotic and Anticoagulant Activities

DS is administered intravenously, just before the shunt circulation is activated. At the conclusion of the test, the thrombus is removed and weighed. Immediately after measuring the thrombus, a portion of the blood is collected at the bottom of the test tube and the time taken to form the first mass on the test tube wall (total clotting time) is measured.

The results of thrombus weight and clotting time are listed in Table 2, which shows that dermatan sulfate exhibits potent antithrombotic action. In this regard, the weight of the thrombus is significantly reduced even with the minimum dose (0.125 mg / kg / intravenous administration). At the same time, the effects on the clotting process are less. In this regard, although the coagulation time gradually increases, it is only statistically and biologically insignificant and requires much higher doses than those effective for antithrombotic action.

TABLE 2

2. Ligation of the Large Vein: Comparison of Different Dosing Methods

DS is administered intravenously 10 minutes prior to ligation, subcutaneously 1 hour prior to ligation, and ileum 15 minutes prior to ligation. At the end of the test, the thrombus is removed and weighed.

The results are listed in Table 3, which shows that DS, when administered intravenously, subcutaneously or ileum, has the effect of inhibiting thrombosis induced by colon ligation of rats. Preliminary evaluations give a dose-effect curve, where the ratio of subtend area is 1: 4: 16 when administered at three defined times, ie, intravenously, subcutaneously, and ileum.

TABLE 3

The present invention also relates to pharmaceutical formulations (vials, tablets, capsules, ointments, syrups, suppositories, dropping agents, etc.) containing a defined amount of dermatan sulfate useful for the treatment of surface and deep thrombosis.

Examples of formulations are as follows.

Capsules containing -20-50-100-200 mg DS

Tablets containing -20-50-100-200mg DS and excipients, deagglomerates and the like commonly used in the pharmaceutical field

Vials containing -20-50-100 mg of DS and aqueous excipients:

Drops containing -20-50-100-200mg / ml DS and aqueous excipients, preservatives, etc. commonly used in the pharmaceutical field

Suppositories containing -20-50-100-200 mg of DS and excipients commonly used in the pharmaceutical field:

Ointment containing -20-50-100-200 mg / g DS and excipients commonly used in the pharmaceutical field

Practical aspects of the manufacturing process are described below to make it easier to reproduce the manufacturing process according to the present invention.

Example 1

10,000 kg of the small intestine mucosa of frozen cattle and pigs are micronized and transferred into a reactor containing 5000 L of 0.01 M CaCl ₂ aqueous solution.

The mass is stirred at 70 rpm until complete homogenization, then the pH is adjusted to 7 with Ca (OH) ₂ solution and heated to 45 °.

10 kg of alkaliase homogenate in 50 l of water is added and the mixture is kept at 45 ° C. for 12 h with continuous stirring. Hydrochloric acid is then added to the flowable mass until it reaches pH 4 and then heated for 30 minutes.

The solution is filtered through a filter press made of celite, the transparent solution is transferred into the reactor and then filtered through a press equipped with a purification card. About 12,000 liters of total clear solution is obtained, and to this solution is added little by little with stirring 10 liters of dimethyl-ethyl-cetyl ammonium ethylsulfate dissolved in 100 liters.

After standing for 6 hours, the clear solution was poured gently and centrifuged at 10000 rpm with a Toniatti centrifuge to recover the residue.

The collected complex is poured into 1000 l of a 2M CaCl ₂ solution containing 100 l of ethanol. Saturated Ca (OH) ₂ solution was added to adjust the pH to about 8 and added to 80 ° C. for about 2 hours with slow stirring. After adding 10 kg of filtration aid, the mixture is filtered through a compressor until the solution is clear. After washing the liquid (cake) 1200 liters) are ultrafiltered through a spiral column (20 cm ² ) with a molecular weight cut-off of 10,000. After concentration to 200 liters, dilute to 1000 liters and again to about 100 liters. This operation is continued until the permeate is actually negative for Ca salt.

30 liters of acetone is added to the solution to precipitate the DS. The precipitate is collected, washed with acetone and dried.

Yield: 8 kg in the form of anhydrous powder

The powder is stirred in 80 L of 2M NaCl solution (12%) for 1 hour, after which the solution is diluted to 150 liters, filtered and precipitated by addition of 45 liters of anhydrous acetone.

The precipitate is collected by compression filter, washed with 40% acetone aqueous solution and dried in vacuo.

Yield: DS sodium salt 7 kg

The powder is dissolved in 100 liters of deionized water, decolorized by passing over anionic resin, depyrified overnight under high temperature conditions, precipitated with acetone in a ratio of 1: 1, filtered and lyophilized.

Yield: DS 4kg with the following characteristics

Molecular weight 35,000

-[α] ₀ -65 °

Ratio of sulfate group to carboxyl group 1.25

-The ratio of uronic acid to galactosamine 1: 1

Single band during electrophoresis analysis

Example 2

Obtained from bovine pancreas and lung, acetone-treated powder to 1000kg and a second micronized 0.01M CaCl ₂ aqueous solution, using a 0.01M CaCl ₂ solution is diluted with 20,000 liters of volume.

After mixing for about 30 minutes, it is maintained at 45 ℃.

The same procedure as described in Example 1 is performed.

Yield: DS 5kg with the following characteristics

Molecular weight 40,000

-[α] ₀ -65 °

Ratio of sulfate group to carboxyl group 1.2

The ratio of uronic acid to galactosamine 1: 1

Single band for electrophoresis analysis

The procedure and results are the same when using the powder of the engine obtained by spray-drying as starting material.

Example 3

100 kg of the frozen fresh animal organs (lungs, blood vessels and small intestine) are ground to obtain pulp and placed into a reactor containing 50 l of 0.01 m CaCl ₂ solution.

The mass is stirred until complete homogenization, then the pH is adjusted to 7.2 with Ca (OH) ₂ and heated to 50 ° C.

200 L of enzyme solution containing 0.05% by weight of superlase was heated to 50 ° C. and then added.

The mixture is stirred at 50 ° C. for 8 hours. After this time the decomposition is complete. The mixture is acidified to pH 5 with HCI, heated to 80 ° C. and filtered under high temperature conditions using filter ground with a filter aid.

The filtrate sample is analyzed, in particular 10 ml of quaternary ammonium: filtrate to be used in the next step is concentrated to 1 ml and then distilled water is added to make a total volume of 2 ml. Separately prepare a 3% agarose plate for electrophoresis in a buffer solution consisting of barium acetate and acetic acid. During the experiment, 5-10 μl of the sample is placed on a plate and primary electrophoresis is performed at 15 volts / cm (about 120 volts). Secondary electrophoresis is then performed in propanediamine buffer solution under the same conditions and then the plate is dried with an infrared source. Stain with 2% toluidine blue cow and bleach with 5% acetic acid.

At this point a photodensitometic reading is performed and the individual MPS area is measured by comparison with the reference standard.

Analytical measurements of MPS showed a total of 200 g and a dermatanthate content of 50 g. 200 g total.

Based on the analytical data, 200 g of hyamine is added to the filtrate.

It is stirred at 70 ° C. for 1 hour, diluted with deionized water, and the water concentration of the solution is adjusted to 0.4M.

It is left for 5 hours and the precipitate in the form of a mixture of quaternary ammonium and the MPS complex is recovered by centrifugation.

This buttery precipitate is first washed with water to remove excess salt, and then 1 liter of acetone is added while maintaining the suspension at ambient temperature under stirring for 3 hours, to selectively dissolve the dermatan sulfate complex. The suspension is centrifuged to give 1 liter of a weak DS solution, to which 2 liters of 3MNaCl solution is added, followed by 2 liters of acetone, and the precipitated DS is separated by centrifugation, washed with ethanol and dried.

In this way, 50 g of DS having the following characteristics is obtained.

Molecular weight 35.000

-[α] ₀ -60 °

Ratio of sulfate group to carboxyl group 1.5

-The ratio of uronic acid to galactosamine 1: 1

Single band for electrophoresis analysis

Example 4

Selective dissolution of the DS complex obtained by treating the MPS mixture as described in Example 3 disperses the resulting solids in 500 ml of distilled water to remove acetone residues, with centrifugation to remove water as well.

The insoluble precipitate is treated with 1.8 liters of ethanol and the resulting suspension is kept at 50 ° C. under stirring for 3 hours to selectively dissolve the heparin sulfate complex.

The subsequent procedure for obtaining heparan sulphate is carried out as described for dermatan sulphate in Example 3.

60 g of heparan sulfate having the following characteristics are obtained.

Molecular weight 30,000

[α] ₀ + 55 °

Single bend during electrophoresis analysis

Example 5

The heparan sulfate complex obtained by treatment as described in Example 4 was selectively dissolved to dissolve the resulting solid material in 3 liters of distilled water to remove the ethanol residue and then centrifuged.

The insoluble precipitate is treated with 5 liters of methanol and the resulting suspension is kept at 50 ° C. under stirring for 3 hours to dissolve the heparin complex.

Subsequent procedures for obtaining and purifying heparin are performed as described for the DS of Example 3.

In this way, 50 g of pharmaceutically pure heparin is obtained.

Claims (17)

a) freezing the organs of fresh animals intact or in the form of powders, b) degrading a stable raw material containing MPS to a homogenate containing aqueous solution of CaCl _{2 with} proteolytic enzymes at alkaline pH and low temperature, d E) acidify the digest, heat and filter, e) treat the filtrate with a quaternary ammonium salt capable of complexing with it to selectively precipitate DS alone or all MPS and f) from the ammonium complex containing DS Recovering and purifying, from the organ of an animal containing a large amount of mucopolysaccharide (MPS) to produce a pharmaceutical purity dermatan sulfate (DS). The method of claim 1, wherein the ultra-micronization is performed by mixing the frozen organ material with 0.01 M CaCl ₂ aqueous solution in a weight ratio of 1: 1. The method of claim 1, wherein the ultrafine microorganism is frozen and the 1M CaCl ₂ aqueous solution is mixed in a weight ratio of 1: 1, and the mass is filtered by cold acetone and finally dried. The method according to claim 1, wherein the ultrafine powder is frozen and the 0.1M CaCl ₂ aqueous solution is mixed at a weight ratio of 1: 1, and the obtained mixture is spray-dried with air at a temperature of less than 150 ° C. The method of claim 1, wherein the ultrafine powder is frozen and the 1M CaCl ₂ aqueous solution containing 10% sorbitol is mixed at a weight ratio of 1: 1, and the obtained mixture is lyophilized. The method of claim 1, wherein the degradation by proteolytic enzymes is carried out by adding the enzyme to an ultra-micronized mixture diluted with water, adjusting the pH to 7-9 and heating to 40-55 ° C. 7. The method of claim 6, wherein the enzyme is added in a weight ratio of raw material: enzyme of 1: 0.0001 to 1: 0.001. The method of claim 6, wherein the pH is adjusted to 7-9 using Ca (OH) ₂ . The method of claim 1, wherein the degradation product is acidified to pH 3-6 and heated to 70-90 ° C. 7. The method of claim 9, wherein the degradation product is acidified using HCL. The method of claim 1, wherein the filtered digest is treated with dimethyl-ethyl-cetylammonium ethylsulfate to selectively precipitate the DS complex. The MPS present according to claim 1 is treated by treating the filtered digest with a quaternary ammonium salt selected from the group consisting of hyamine, cetyl-trimethyl ammonium bromide and cetyl-dimethyl-ethyl ammonium bromide at a temperature of 60 to 70 ° C. Precipitation in the form of a complex. The method according to claim 12, wherein the DS complex compound is selectively dissolved using acetone. The method of claim 11, wherein the DS is recovered from the ammonium complex by treating the ammonium complex with a 10% ethanol-containing 2M CaCl ₂ aqueous solution, adjusting the pH to 7-9, and then heating to 60-80 ° C. 13. The method of claim 13, wherein the DS is recovered from the ammonium complex by treating the ammonium complex with a 3M salt solution in an amount such that the volume ratio of the acetone extract to the salt solution is 0.25 to 1. The method according to claim 12 or 13, wherein the residue produced by treatment with acetone is treated with ethanol to selectively dissolve the heparan sulfate complex compound. 17. The method of claim 16, wherein the residue produced by treatment with ethanol is treated with methanol to selectively dissolve the heparin complex.